Long-lasting silencing of orexin/hypocretin neurons using
archaerhodopsin induces slow-wave sleep in mice
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Citation
Tsunematsu, Tomomi, Sawako Tabuchi, Kenji F. Tanaka,
Edward S. Boyden, Makoto Tominaga, and Akihiro Yamanaka.
“Long-Lasting Silencing of Orexin/hypocretin Neurons Using
Archaerhodopsin Induces Slow-Wave Sleep in Mice.”
Behavioural Brain Research 255 (October 2013): 64–74.
As Published
http://dx.doi.org/10.1016/j.bbr.2013.05.021
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Elsevier
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Final published version
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Thu May 26 09:15:49 EDT 2016
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http://hdl.handle.net/1721.1/90302
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Behavioural Brain Research 255 (2013) 64–74
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Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Research report
Long-lasting silencing of orexin/hypocretin neurons using
archaerhodopsin induces slow-wave sleep in mice夽
Tomomi Tsunematsu a,b , Sawako Tabuchi a,b,c , Kenji F. Tanaka d , Edward S. Boyden e ,
Makoto Tominaga a,c , Akihiro Yamanaka a,f,g,∗
a
Division of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
The Japan Society for the Promotion of Sciences, Tokyo 102-8472, Japan
c
Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki 444-8787, Japan
d
Department of Neuropsychiatry, School of Medicine, Keio University, Tokyo 160-8582, Japan
e
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
f
Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan
g
PRESTO, JST, Kawaguchi 332-0012, Japan
b
h i g h l i g h t s
•
•
•
•
New transgenic mice, TetO archaerhodopsin (ArchT) BAC mice, were generated.
Specific expression of ArchT was confirmed by breeding with orexin-tTA mice.
Slice patch clamp confirmed that green light illumination silenced orexin neurons.
One hour silencing of orexin neurons induced slow-wave sleep in the dark period.
a r t i c l e
i n f o
Article history:
Received 17 December 2012
Received in revised form 8 April 2013
Accepted 14 May 2013
Available online 21 May 2013
Keywords:
Orexin
Hypocretin
Optogenetic
Archaerhodopsin
Slow-wave sleep
a b s t r a c t
Orexin/hypocretin neurons have a crucial role in the regulation of sleep and wakefulness. Recent optogenetic studies revealed that the activation or inhibition of orexin neuronal activity affects the probability
of sleep/wakefulness transition in the acute phase. To expand our understanding of how orexin neurons
maintain wakefulness, we generated new transgenic mice in which orexin neurons expressed archaerhodopsin from Halorubrum strain TP009 (ArchT), a green light-driven neuronal silencer, using the tet-off
system (orexin-tTA; TetO ArchT mice). Slice patch clamp recordings of ArchT-expressing orexin neurons
demonstrated that long-lasting photic illumination was able to silence the activity of orexin neurons.
We further confirmed that green light illumination for 1 h in the dark period suppressed orexin neuronal
activity in vivo using c-Fos expression. Continuous 1 h silencing of orexin neurons in freely moving orexintTA; TetO ArchT mice during the night (the active period, 20:00–21:00) significantly increased total time
spent in slow-wave sleep (SWS) and decreased total wake time. Additionally, photic inhibition increased
sleep/wakefulness state transitions, which is also evident in animals lacking the prepro-orexin gene,
orexin neurons, or functional orexin-2 receptors. However, continuous 1 h photic illumination produced
little effect on sleep/wakefulness states during the day (the inactive period, 12:00–13:00). These results
suggest that orexin neuronal activity plays a crucial role in the maintenance of wakefulness especially in
the active phase in mice.
© 2013 The Authors. Published by Elsevier B.V. All rights reserved.
1. Introduction
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits
non-commercial use, distribution, and reproduction in any medium, provided the
original author and source are credited.
∗ Corresponding author at: Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan.
Tel.: +81 52 789 3864; fax: +81 52 789 3889.
E-mail address: yamank@riem.nagoya-u.ac.jp (A. Yamanaka).
Orexin A and B (or hypocretin-1 and hypocretin-2) are a pair
of neuropeptides expressed in a specific population of neurons
in the lateral hypothalamic area (LHA) [1,2]. Animals that lack
the orexin peptide (prepro-orexin knockout mice), orexin neurons
(orexin/ataxin-3 transgenic mice), or functional orexin 2 receptors
(OX2Rs, via OX2R knockout mice and OX2R mutated dogs) show
phenotypes remarkably similar to those of the human sleep disorder, narcolepsy [3–6]. Narcoleptic patients show symptoms of
0166-4328/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbr.2013.05.021
T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
fragmented sleep and wakefulness, sleep-onset rapid eye movement (REM), and cataplexy, a sudden bilateral postural muscle
weakness usually precipitated by emotional stimuli. In addition,
the concentration of orexin-A (hypocretin-1) peptide in the cerebrospinal fluid (CSF) of narcoleptic patients is greatly reduced
[7], and a specific degeneration of orexin neurons has also been
reported [8,9]. Indeed, central administration of orexin induces
wakefulness and decreases slow-wave sleep (SWS) and REM sleep
durations [10,11]. Orexin neurons are thought to be phasically
active during wakefulness and silent during SWS [12–14]. These
findings indicate that the orexin system plays a crucial role in the
regulation of sleep/wakefulness, especially in the maintenance of
arousal.
Orexin neurons project throughout the brain and densely innervate monoaminergic nuclei, including the dorsal raphe nucleus
(DR), locus coeruleus (LC) and tuberomammillary nucleus (TMN)
[1,15,16]. These monoaminergic nuclei are implicated in increasing
arousal and promoting wakefulness. The activity of monoaminergic
neurons in the DR, LC and TMN is synchronized and strongly associated with sleep/wakefulness states [17–20]. That is, these neurons
exhibit tonic firing during wakefulness, decreased firing during
SWS, and electrical quiescence during REM sleep. Electrophysiological experiments have shown that serotonergic cells in the DR,
noradrenergic cells in the LC and histaminergic cells in the TMN are
activated by orexin via the activation of orexin 1 receptors (OX1Rs)
and/or OX2Rs [21–24]. On the other hand, GABAergic neurons in
the ventrolateral preoptic area (VLPO) seem to play a crucial role
in sleep initiation and maintenance. Neurons in the VLPO exhibit
high frequency firing during sleep, whereas they display less firing during wakefulness [25–27]. Orexin neurons are innervated by
GABAergic neurons in the VLPO [28], and are strongly hyperpolarized by muscimol or baclofen, a GABAA receptor agonist or a GABAB
receptor agonist, respectively [29,30]. Sleep-promoting GABAergic neurons in the VLPO and arousal-promoting monoaminergic
neurons in the DR, LC and TMN project reciprocally and mutually
inhibit each other’s activity [31]. As such, these neuronal circuitries
are important for the regulation of sleep/wakefulness. In particular,
orexin neurons have a crucial role in the maintenance of wakefulness by modulating the mutual inhibition because elimination of
orexin neurons results in fragmentation of sleep/wakefulness as
displayed in narcolepsy.
Recent studies using optogenetics have demonstrated the
importance of orexin neuronal activity in the regulation of
sleep and wakefulness states. Activation of orexin neurons via
channelrhodopsin-2 (ChR2), a blue light-activated, non-selective
cation channel, or melanopsin, a blue light-activated Gq type of Gprotein coupled receptor, increases the probability of transitions to
wakefulness from SWS and REM sleep in mice [32–34]. In contrast,
acute inhibition of orexin neurons via halorhodopsin (HaloR), an
orange light-driven chloride pump, promotes the transition to SWS
from wakefulness [35]. These results directly establish a causal link
between activity of orexin neurons and sleep/wakefulness transition. However, technical limitations of the first generation of
optogenetics make it difficult to control the activity of neurons for
extended periods.
To address this, we generated new transgenic mice in which
orexin neurons express Archaerhodopsin T (ArchT), a green lightdriven powerful neuronal silencer. ArchT was recently discovered
from Halorubrum strain TP009 and forms proton pumps suitable
for complete silencing [36]. Using these mice, we were able to continuously inhibit orexin neurons in vitro and in vivo. We found that
in vivo inhibition of orexin neurons for 1 h increased time spent in
SWS and decreased time spent in wakefulness only during the dark
period. This result suggests that orexin neuronal activity plays an
important role in the maintenance of wakefulness, especially in the
active phase.
65
2. Methods
2.1. Animal usage
All experimental procedures involving animals were approved by the National
Institute for Physiological Sciences Animal Care and Use Committee and were in
accordance with NIH guidelines. All efforts were made to minimize animal suffering
or discomfort and to reduce the number of animals used.
2.2. Generation of TetO ArchT BAC transgenic mice
To generate the TetO ArchT BAC transgenic mice, an archaerhodopsin cDNA
fragment from Halorubrum strain TP009 (ArchT) [36] was swapped with that
of the ChR2(C128S) sequence in TetO ChR2(C128S)EGFP-Neo construct flanked on
both sides by a ␤-actin gene homology arm [37]. BAC DNA (clone RP23-289L7)
was modified by BAC recombination with this transgene with homology arms,
resulting in the insertion of TetO ArchT-EGFP-Neo cassette 30 bp downstream of
a mouse ␤-actin polyA signal. To remove the FRT-flanked Neo cassette, recombined BAC DNA was electroporated into strain SW105 (kindly gifted from Dr. Neal
Copeland, National Cancer Institute) that possesses flippase activity. Linearized
modified BAC DNA was injected into fertilized eggs from C57BL/6J (Clea-Japan
Inc., Tokyo, Japan) mice. Founders were bred with C57BL/6J mice to produce
stable TetO ArchT transgenic lines. A total of 3 TetO ArchT transgene-positive
founders were obtained. We crossed these mice with orexin-tTA mice [38] to obtain
orexin neuron-specific ArchT-EGFP-expressing mice (see Fig. 1). Immunohistochemical analysis of the N1 generation revealed that line A showed the highest
EGFP expression (see Fig. 2). Thus, line A was used for further experimentation.
2.3. Brain slice preparation
Male and female orexin-tTA; TetO ArchT double transgenic mice (3–4 weeks old)
were used for whole cell recordings. The mice were deeply anesthetized with isoflurane (Abbott Japan, Tokyo, Japan) and decapitated. Brains were quickly isolated
in ice-cold cutting solution consisting of (in mM): 280 sucrose, 2 KCl, 10 HEPES,
0.5 CaCl2 , 10 MgCl2 , 10 glucose, pH 7.4 with NaOH, bubbled with 100% O2 . Brains
were cut coronally into 350 ␮m slices with a microtome (VTA-1200S, Leica, Wetzlar, Germany). Slices containing the LHA were transferred to an incubation chamber
shielded from light and filled with a physiological solution containing (in mM): 135
NaCl, 5 KCl, 1 CaCl2 , 1 MgCl2 , 10 HEPES, 10 glucose, pH 7.4 with NaOH, bubbled with
100% O2 and incubated for at least 1 h at room temperature (RT; 24–26 ◦ C).
2.4. Electrophysiological recordings
At RT, the slices were transferred to a recording chamber (RC-27L, Warner
Instrument Corp., CT) on a fluorescence microscope stage (BX51WI, Olympus, Tokyo,
Japan). Neurons having EGFP fluorescence were identified as orexin neurons and
were subjected to electrophysiological recordings. The fluorescence microscope was
equipped with an infrared camera (C2741-79, Hamamatsu Photonics, Hamamatsu,
Japan) for infrared differential interference contrast (IR-DIC) imaging and a cooled
charge-coupled device (CCD) camera (Cascade 650, Roper Scientific, Tucson, AZ) for
fluorescent imaging. Each image was displayed separately on a monitor and saved
on a computer. Recordings were carried out with an Axopatch 200B amplifier (Axon
Instruments, Foster City, CA) using a borosilicate pipette, (GC150-10, Harvard Apparatus, Holliston, MA) prepared by a micropipette puller (P-97, Sutter Instruments,
Novato, CA), and filled with intracellular solution (4–6 M) consisting of (in mM):
138 K-gluconate, 10 HEPES, 8 NaCl, 0.2 EGTA-Na3 , 2 MgATP, 0.5 Na2 GTP, pH 7.3 with
KOH. The osmolality of the solution was checked by a vapor pressure osmometer
(model 5520, Wescor, Logan, UT). The osmolality of the internal and external solutions was 280–290 and 320–330 mOsm/L, respectively. The liquid junction potential
of the patch pipette and perfused extracellular solution was estimated to be 16 mV
and was corrected in the data. Recording pipettes were under positive pressure
while advancing toward individual cells in the slice, and tight seals on the order
of 1.0–1.5 G were made by negative pressure. The membrane patch was then
ruptured by suction. The series resistance during recording was 10–25 M. The
reference electrode was an Ag–AgCl pellet immersed in bath solution. During recordings at RT, cells were superfused with extracellular solution at a rate of 1.6 mL/min
using a peristaltic pump (Dynamax, Rainin, Oakland, CA). Green light (549 ± 7.5 nm)
was generated by lumencor (SPECTRA light engine, Lumencor. Inc., OR, USA).
The output signal was low-pass filtered at 5 kHz and digitized at 10 kHz. Data
were recorded on a computer through a Digidata 1322A A/D converter using pClamp
software version 10.2 (Axon Instruments, Union City, CA).
2.5. Drugs
Tetrodotoxin (TTX) (Wako, Osaka, Japan) was dissolved in extracellular solution at a concentration of 1 ␮M. During the experiments, TTX was applied by bath
application.
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T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
Fig. 1. Generation of orexin-tTA; TetO ArchT double-transgenic mouse. (A) Structure of TetO ArchT BAC transgene. There is the sequence of tetracycline operater (TetO, yellow
square), which binds tTA protein, and minimal promoter (P, circle) in 0.7 kb upstream of ArchT-EGFP (light blue square). The ␤-actin gene consists of 6 exons (blue squares), and
the TetO ArchT cassette is inserted just downstream of the polyadenylation signal of the ␤-actin gene. (B) Schematic illustration of the tetracycline-controlled gene expression
system and tTA-induced ArchT expression in orexin neurons. (C) Schema indicating how orexin-tTA mice are bred with TetO ArchT mice to generate double-transgenic mice,
orexin-tTA; TetO ArchT mice. hOX promoter, human prepro-orexin promoter. P, minimal promoter. TetO, tetracycline operator. tTA, tetracycline-controlled transcriptional
activator. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.6. Immunohistochemistry
Male and female orexin-tTA; TetO ArchT transgenic mice (6 weeks old) were
deeply anesthetized with isoflurane and perfused sequentially with 20 mL of chilled
saline and 20 mL of chilled 10% formalin solution (Wako). The brains were removed
and immersed in the above fixative solution for 24 h at 4 ◦ C and then immersed
in a 30% sucrose solution for at least 2 days. The brains were quickly frozen in
embedding solution (Sakura Finetechnical Co. Ltd., Tokyo, Japan). For orexin and GFP
double-staining, coronal sections (40 ␮m) of orexin-tTA; TetO ArchT transgenic mice
brains were incubated with mouse anti-GFP antiserum (1/1000, Wako) for 24 h at
4 ◦ C. These sections were incubated with Alexa 488-labeled donkey anti-mouse IgG
(1/1000, Biotium, CA, USA) for 1 h at RT. The sections were then incubated with goat
anti-orexin antiserum (1/1000, Santa Cruz Biotechnology, CA, USA) for 24 h at 4 ◦ C
and incubated with Alexa 594-labeled donkey anti-goat IgG (1/1000, Biotium) for
1 h at RT. The sections were mounted and examined with a fluorescence microscope
(BZ-9000, Keyence, Osaka, Japan).
For orexin and c-Fos double staining, coronal sections (40 ␮m) were stained
by the avidin-biotin-peroxidase method. Brain sections were incubated for 40 min
in phosphate-buffered saline (PBS) containing 0.3% H2 O2 to inactivate endogenous
peroxidase. Sections were transferred into PBS containing 0.25% Triton X-100 and
1% bovine serum albumin fraction V (PBS-BX) for 30 min and then incubated with
rabbit anti-c-Fos antibody (AB5, Calbiochem, Billerica, MA, USA) diluted 1/6000 in
PBS-BX overnight at 4 ◦ C. Sections were then incubated with biotin labeled antirabbit IgG goat antibody (Vector Laboratories, Burlingame, CA) for 1 h at RT followed
by incubation with avidin and biotinylated peroxidase complex solution for 1 h at
RT. Bound peroxidase was visualized by incubating sections with 0.01 M imidazole
acetate buffer containing 0.05% 3,3-diaminobenzidine tetrahydrochloride (Dako,
Carpinteria, CA), 0.0015% H2 O2 and 2.5% nickel ammonium sulfate, which resulted
in a black reaction product. For double-labeling, sections were incubated overnight
at 4 ◦ C with guinea pig anti-orexin antibody diluted 1/800 in PBS-BX. Sections
were then incubated with biotin labeled anti-guinea pig IgG goat antibody (Vector
Laboratories) for 1 h at RT. Bound peroxidase was visualized by DAB-buffer tablet
(Merck, NJ) with 0.0015% H2 O2 , resulting in a golden-brown reaction product.
The sections were mounted and examined with a microscope (BZ-9000, Keyence)
or a confocal microscope (LSM710, Zeiss, Germany). To confirm the specificity of
antibodies, incubations without primary antibody were conducted as a negative
control in each experiment and in each case no signal was observed.
2.7. In vivo photo illumination using freely moving mice
15-Week-old male orexin-tTA; TetO ArchT transgenic mice were housed under
controlled lighting (12 h light/dark cycle; lights on from 8:00 to 20:00) and temperature (22 ◦ C) conditions. Food and water were available ad libitum. Mice were
anesthetized with isoflurane using a vaporizer for small animals (400, Bio Research
Center Co. Ltd., Nagoya, Japan) and positioned in a stereotaxic frame (David Kopf
Instruments, Tujunga, CA). Plastic fiber optics (0.5 mm in diameter; Eska, Mitsubishi
Rayon Co. Ltd., Tokyo, Japan) were bilaterally implanted into the hypothalamus
approximately 1 mm above the LHA (1.2 mm posterior, 1 mm lateral from bregma,
3.5 mm depth from brain surface). Electrodes for EEG and EMG were implanted on
the skull and neck muscle, respectively. These were attached to the skull using dental cement (GC Corporation, Tokyo, Japan). The mice were then housed separately
for a recovery period of at least 7 d.
Continuous EEG and EMG recordings were carried out through a slip ring
(Air Precision, Le Pressis Robinson, France) designed so that the movement of
the mouse was unrestricted. EEG and EMG signals were amplified (AB-610J,
Nihon Koden, Tokyo, Japan), filtered (EEG, 1.5–30 Hz; EMG, 15–300 Hz), digitized
at a sampling rate of 128 Hz, and recorded using SleepSign software version 3
(Kissei Comtec, Nagano, Japan). Green light (542 ± 13.5 nm) was generated by
Lumencor and applied through plastic optical fibers bilaterally inserted 1 mm
above the LHA. An optical swivel (COME2, Lucir, Tsukuba, Japan) was used for
unrestricted in vivo photo illumination. Green light power intensity at the tip of the
plastic fiber optics (0.5 mm diameter) was 23 mW/mm2 as measured by a power
T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
67
Fig. 2. ArchT is specifically expressed in orexin neurons in the orexin-tTA; TetO ArchT double-transgenic mouse brain. (A–C) Immunohistochemical analyses revealed the
expression rate of two lines of TetO ArchT transgenic mice, line A (A) and line B (C). Orexin-IR neurons located in the LHA (Alexa594, red). GFP-IR neurons, indicating ArchT
expression, are also observed in the LHA (Alexa488, green). The merged image shows specific expression of ArchT in orexin neurons in the LHA of orexin-tTA; TetO ArchT
transgenic mice (upper panels, scale bar, 300 ␮m). In line A (A), 72.4 ± 12.2% (n = 4) of orexin neurons express ArchT. In line B (C) 23.9 ± 2.2% (n = 3) of orexin neurons express
ArchT. No expression of ArchT outside of orexin neurons was observed throughout the brain in either transgenic mouse line. The lower panels are higher magnifications of
the squared region in the upper panels (scale bar, 50 ␮m). (B) Confocal microscopic image of orexin-tTA; TetO ArchT (line A) mouse showing that ArchT expression is observed
in the soma and dendrites of orexin neurons. Arrows indicate an example of GFP-expressing orexin neurons. Arrowheads indicate an example of GFP-non-expressing orexin
neurons. Scale bar, 10 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
meter (VEGA, Ophir Optronics Ltd., Wilmington, MA). Each animal’s behavior was
monitored through a CCD video camera and recorded on a computer synchronized
with EEG and EMG recordings using the SleepSign video option system (Kissei
Comtec). The infrared activity monitor was a sensor mounted on top of the
cage to measure locomotor activity. The sensor’s output signals representing the
magnitude of each animal’s movement were digitally converted and transferred to a
computer.
Polysomnographic recordings were automatically scored offline, with each
epoch scored as wakefulness, SWS, or REM sleep by SleepSign according to standard
criteria [24,39]. All vigilance state classifications assigned by SleepSign were examined visually and corrected if necessary. To score sleep/wakefulness fragmentation
during light illumination, the number of changes in sleep/wakefulness state, such
as the transition from wakefulness to SWS, from SWS to REM sleep, from SWS to
wakefulness, and from REM sleep to wakefulness, were counted as state transitions.
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T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
2.8. Statistical analysis
Data were analyzed by paired t-test, un-paired t-test, one-way ANOVA, or twoway ANOVA as appropriate for the parameters examined using KaleidaGraph 4.0
software (Hulinks, Tokyo, Japan). When appropriate, ANOVA tests were followed by
post hoc analysis of significance using Fisher’s Protected Least Significant Difference
test. Probability (p)-values less than 0.05 were considered statistically significant.
3. Results
3.1. Specific expression of ArchT in orexin neurons
TetO ArchT BAC transgenic mice were generated in which
ArchT-EGFP fusion protein was induced by using the tetracyclinecontrolled gene induction system. To achieve sufficient ArchT
expression to make targeted cells hyperpolarize, we took advantage
of the enhancing gene expression module; the TetO cassette was
inserted downstream of ␤-actin gene allele, a housekeeping gene,
in the BAC (Fig. 1A) [37]. A total of three TetO ArchT BAC transgenepositive founders were obtained (lines A–C). The transgene was
transferred to the N1 generation of lines A and B, but not line C.
tTA expression is driven by a specific promoter, in this case the
human prepro-orexin promoter, resulting tTA expression in orexin
neurons. Subsequently, tTA transactivates ArchT-EGFP induction by
binding TetO sequences in orexin neurons (Fig. 1B). TetO ArchT mice
(line A or B) were bred with orexin-tTA mice and orexin-tTA; TetO
ArchT double-transgenic mice were used in this study (Fig. 1C).
The specific expression of ArchT (lines A and B) in orexin neurons was confirmed by double-labeled immunohistochemistry. An
anti-GFP antibody was used to detect ArchT-EGFP fusion proteins
in double-transgenic mice. Merged pictures (GFP-IR and orexin-IR)
revealed that ArchT-EGFP was exclusively observed in orexin neurons of orexin-tTA; TetO ArchT (line A) (Fig. 2A and B) and orexin-tTA;
TetO ArchT (line B) (Fig. 2C) mice. In both double-transgenic lines,
we did not observe ArchT-EGFP expressing cells other than orexin
neurons. Confocal microscopic observation revealed that ArchT was
detected in the soma and dendrites of orexin neurons. Orexin neurons expressing ArchT did not show blebbing or other features
indicative of inappropriate trafficking (Fig. 2B). The number and
morphology of orexin-IR neurons in orexin-tTA; TetO ArchT (line
A) transgenic mice were indistinguishable from those wild-type
mice or monogenic littermate mice (orexin-tTA or TetO ArchT mice,
data not shown), suggesting that ArchT expression is not toxic to
orexin neurons. ArchT expression rates (GFP-IR/orexin-IR × 100%)
in orexin-tTA; TetO ArchT (line A) and orexin-tTA; TetO ArchT (line B)
mice was 72.4 ± 12.2% (n = 4) and 23.9 ± 2.2% (n = 3), respectively.
TetO ArchT (line A) mice were used for the following in vitro and
in vivo experiments since line A showed a significantly higher gene
expression rate.
3.2. Green light illumination inhibits the activity of
ArchT-expressing orexin neurons
To confirm the function of ArchT expressed in the transgenic mouse brain, slice patch clamp analyses were performed.
Orexin neurons were whole cell current-clamped and green light
(549 ± 7.5 nm) was illuminated, since previous reports revealed
that ArchT exhibited the most potent sensitivity for yellow-green
light illumination (around 570 nm) [36]. Green light was illuminated through an objective lens. Green light illumination (10%
light intensity; 2.5 mW) instantaneously hyperpolarized the membrane potential and completely inhibited the generation of action
potentials in orexin neurons for 30 s (Fig. 3A). After light cessation, the activity in silenced orexin neurons recovered ultimately
to baseline firing rates without showing a rapid rebound activity.
The magnitude of ArchT-mediated hyperpolarization depended on
the light intensity in the presence of TTX (Fig. 3B). Green light
illumination induced transient currents and sustained currents
and these two were separately analyzed. Green light-induced
hyperpolarizations immediately after light activation of orexin
neurons (transient potential) were 4.1 ± 1.6 mV (n = 8, p = 0.77, not
significantly different (NS), ANOVA), 20.7 ± 4.1 mV (n = 8, p = 0.08,
NS, ANOVA), 35.1 ± 6.3 mV (n = 8, p = 0.002, ANOVA), 38.1 ± 6.7 mV
(n = 8, p = 0.001, ANOVA), 43.4 ± 7.9 mV (n = 8, p < 0.001, ANOVA),
60.7 ± 9.8 mV (n = 10, p < 0.001, ANOVA), 72.2 ± 11.8 mV (n = 10,
p < 0.001, ANOVA), 70.4 ± 11.8 mV (n = 10, p < 0.001, ANOVA) and
69.6 ± 11.7 mV (n = 10, p < 0.001, ANOVA) under light intensities of
1, 2, 3, 4, 5, 10, 25, 50 and 100% (25 mW), respectively (Fig. 3C).
The magnitude of light-induced hyperpolarization showed a gradual decrease during light illumination. The hyperpolarizations
just before light cessation (sustained) were 2.9 ± 1.1 mV (n = 8,
p = 0.85, NS, ANOVA), 17.3 ± 4.0 mV (n = 8, p = 0.14, NS, ANOVA),
24.8 ± 4.4 mV (n = 8, p = 0.03, ANOVA), 26.1 ± 4.0 mV (n = 8, p = 0.02,
ANOVA), 29.5 ± 4.7 mV (n = 8, p = 0.01, ANOVA), 42.8 ± 7.3 mV
(n = 10, p < 0.001, ANOVA), 49.1 ± 8.0 mV (n = 10, p < 0.001, ANOVA),
49.1 ± 9.4 mV (n = 10, p < 0.001, ANOVA) and 48.7 ± 10.1 mV (n = 10,
p < 0.001, ANOVA) under light intensities of 1, 2, 3, 4, 5, 10, 25, 50
and 100% (25 mW), respectively (Fig. 3C). In contrast, green light
(100% intensity) had little effect on the membrane potential of
EGFP-negative neurons in orexin-tTA; TetO ArchT mice (0.8 ± 0.9 mV,
n = 9).
Under voltage clamp mode, green light was illuminated onto
ArchT-expressing orexin neurons in the presence of TTX. At the
holding membrane potential of −60 mV, green light illumination
induced outward currents in a light intensity-dependent manner (Fig. 3D). Green light-induced transient outward currents
were 5.0 ± 0.8 pA (n = 11, p = 0.90, NS, ANOVA), 23.7 ± 5.5 pA
(n = 11, p = 0.51, NS, ANOVA), 51.7 ± 11.8 pA (n = 11, p = 0.15, NS,
ANOVA), 67.7 ± 15.6 pA (n = 11, p = 0.06, NS, ANOVA), 80.6 ± 19.0 pA
(n = 11, p = 0.02, ANOVA), 117.0 ± 28.9 pA (n = 12, p < 0.001, ANOVA),
165.7 ± 40.3 pA (n = 12, p < 0.001, ANOVA), 185.6 ± 45.6 pA (n = 12,
p < 0.001, ANOVA), and 201.1 ± 49.7 pA (n = 12, p < 0.001, ANOVA)
under light intensities of 1, 2, 3, 4, 5, 10, 25, 50 and 100%
(25 mW), respectively (Fig. 3E). Sustained outward currents were
4.7 ± 0.8 pA (n = 11, p = 0.91, NS, ANOVA), 19.2 ± 3.8 pA (n = 11,
p = 0.60, NS, ANOVA), 39.0 ± 8.3 pA (n = 11, p = 0.27, NS, ANOVA),
47.8 ± 9.5 pA (n = 11, p = 0.18, NS, ANOVA), 55.8 ± 10.9 pA (n = 11,
p = 0.12, NS, ANOVA), 78.0 ± 15.5 pA (n = 12, p = 0.03, ANOVA),
96.5 ± 17.8 pA (n = 12, p = 0.006, ANOVA), 101.2 ± 17.8 pA (n = 12,
p = 0.004, ANOVA) and 103.2 ± 16.9 pA (n = 12, p = 0.003, ANOVA)
under light intensities of 1, 2, 3, 4, 5, 10, 25, 50 and 100% (25 mW),
respectively (Fig. 3E).
Next, green light-induced inhibition of orexin neurons was confirmed using a loose cell-attached recording, which monitors firing
frequency without affecting the intracellular conditions of the
neuron being recorded. Before light illumination, orexin neurons
exhibited spontaneous firing at a rate of 6.5 Hz. Long-lasting green
light illumination (10%, 2.5 mW) for 10 min completely inhibited
spontaneous firing in ArchT-expressing orexin neurons (Fig. 3F).
Approximately 7 min after light termination, firing had recovered
to basal levels although the neuronal activity remained attenuated
for a few minutes, which was in agreement with the current clamp
results. These results strongly suggest that light illumination of
the hypothalamus would inhibit the activity of orexin neurons in
vivo.
3.3. Inhibition of orexin neuronal activity by green light
illumination in vivo
To confirm whether acute (1 min) silencing of orexin neuronal
activity induces a transition from wakefulness to SWS in orexintTA; TetO ArchT mice, which have previously been described by
our group using orexin/Halo mice [35], we performed in vivo 1-min
T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
69
Fig. 3. Green light inhibits the activity of ArchT-expressing orexin neurons in vitro. (A) Under current-clamp mode, continuous green light (549 ± 7.5 nm) illumination for
30 s induced hyperpolarization and completely inhibited spontaneous action potentials in ArchT-expressing orexin neurons. (B) In the presence of TTX, green light produced
hyperpolarizations in a light intensity-dependent manner. Light intensities were 1, 2, 3, 4, 5, 10, 25, 50 and 100% (23 mW), from left to right. (C) Bar graph summarizing
the data in B. (D) Under whole-cell voltage-clamp mode, with membrane potential holding at −60 mV, green light induced outward currents in a light intensity-dependent
manner in the presence of TTX. (E) Bar graph summarizing the data in D. (F) In a loose cell-attached recording from ArchT-expressing orexin neurons, continuous green light
illumination for 10 min also completely inhibited spontaneous firing. Green light was applied through an objective lens for a duration indicated by the green bars. Values
represent means ± S.E.M. *p < 0.05 vs. 100% green light illumination of EGFP (−) neurons. Transient, transient potential or current was observed immediately after light onset.
Sustained, sustained potential or current was observed just before light cessation. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of the article.)
photic illumination within the hypothalamus using freely moving
mice. To determine sleep/wakefulness states during recording,
mice were chronically implanted with EEG and EMG electrodes.
To deliver green light to the hypothalamus, plastic fiber optics
were bilaterally implanted into the brain. During the time when
mice displayed spontaneous wakefulness in the late light period
(13:00–19:00), green light was illuminated at random for 1 min
through the fiber optics from a light source (542 ± 13.5 nm). During
green light illumination into the hypothalamus, EEG power gradually increased and EMG power gradually decreased, suggesting an
induction of SWS (Fig. 4A). After about 40 s from light on, all orexintTA; TetO ArchT transgenic mice exhibited SWS but not REM sleep
(n = 12, Fig. 4B–D). In contrast, in the dark period (21:00–0:00), during which nocturnal mice have the greatest spontaneous activity
and arousal levels, green light illumination into the hypothalamus
had no effect on sleep/wakefulness patterns in freely-moving
orexin-tTA; TetO ArchT transgenic mice (n = 11, Fig. 4E–H). These
results are in agreement with our previous results [35].
3.4. In vivo green light illumination of the hypothalamus
increases time spent in slow wave sleep during the dark period
but not during the light period
Next, to confirm whether green light illumination could inhibit
the activity of orexin neurons in vivo, double immunohistochemical staining for c-Fos and orexin was performed. To deliver green
light into the hypothalamus, plastic fiber optics were bilaterally
implanted into the brain of orexin-tTA; TetO ArchT mice. Green light
was illuminated for 1 h during the dark period (20:00–21:00), at
the time of day when orexin neuronal activity is maximal. Subsequently, the animals were sacrificed and the brains were fixed at
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T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
Fig. 4. Acute optogenetic inhibition of orexin neurons in vivo induces SWS in orexin-tTA; TetO ArchT mice. EEG and EMG electrodes were chronically implanted to determine
sleep/wakefulness states. Plastic fiber optics (0.5 mm diameter) were bilaterally inserted above the hypothalamus. Green light from a Lumencor (542 ± 13.5 nm) was applied
through the fiber optics for 60 s. (A and E) Representative traces show EEG (upper trace) and EMG (lower trace) during green light illumination during the second half of
the light period (13:00–19:00) (A) and during the dark period (20:00–0:00) (E). Green light illumination was initiated at random when orexin-tTA; TetO ArchT mice had been
awake for at least 8s (2 epochs) during either the light or dark period. Line graphs summarize the percentage of animals exhibiting wakefulness (B and F), SWS (C and G),
and REM sleep (D and H) during green light illumination. Wake; wakefulness. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of the article.)
21:00. Orexin-tTA; TetO ArchT mice without green light illumination
were used as controls.
In the control group (without light illumination), several
double-labeled neurons (orexin-positive neurons with c-Fospositive nuclei) are observed (Fig. 5A, arrowheads). The c-Fos
expression rate (orexin-IR with c-Fos-IR/orexin-IR × 100%) in control mice was 30.5 ± 1.9% (n = 11) (Fig. 5B). Comparatively, the c-Fos
expression rate in the 1 h green light illumination group was significantly decreased to 13.2 ± 1.9% (Fig. 5A and B, n = 10, p < 0.001,
un-paired t-test). These observations demonstrated that continuous green light illumination for 1 h appropriately inhibited the
activity of orexin neurons of orexin-tTA; TetO ArchT mice.
To study the physiological significance of orexin neuronal activity in the regulation of sleep/wakefulness, we performed in vivo
long-lasting green light illumination using freely moving orexintTA; TetO ArchT mice. Based on the results of c-Fos immunostaining,
continuous 1 h green light illumination was applied (Fig. 6A). First,
we examined the effect of green light illumination during the
dark period when nocturnal mice have the greatest spontaneous
activity and arousal levels (20:00–21:00) (Fig. 6B). Orexin-tTA;
TetO ArchT mice without light illumination were used as controls. A typical hypnogram of a control mouse shows that the
mouse spent most of the time awake at the beginning of the
dark period (Fig. 6Ba). In contrast, a mouse receiving green light
illumination frequently transitioned to SWS (Fig. 6Bb). The total
time spent in SWS during green light illumination was significantly increased compared with that of control mice (Fig. 6Bc).
In the control mice, the total time in wakefulness, SWS and REM
sleep over 1 h was 50.7 ± 3.0 min (wakefulness, n = 7), 9.1 ± 3.0 min
(SWS, n = 7) and 0.2 ± 0.1 min (REM, n = 7), respectively. In the
green light-illuminated group, the total time in wakefulness,
SWS and REM sleep over 1 h was 40.7 ± 4.1 min (wakefulness,
n = 7, p = 0.049, paired t-test), 9.1 ± 3.0 min (SWS, n = 7, p = 0.048,
paired t-test) and 0.3 ± 0.2 min (REM, n = 7, p = 0.94, NS, paired
t-test), respectively. These results suggested that inhibition of
orexin neuronal activity resulted in an increased time spent
T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
71
Fig. 5. Inhibition of orexin neurons by green light illumination in vivo. Orexin-tTA; TetO ArchT transgenic mice were decapitated during the dark period, at 21:00. The
light illumination group received green light into the hypothalamus for 1 h (from 20:00 to 21:00) before decapitation. Green light was applied through plastic fiber optics
(diameter of 0.5 mm) that were bilaterally inserted into the hypothalamus. Coronal sections through the hypothalamus containing orexin neurons were studied using double
immunostaining. (A) Immunoreactivity for orexin (brown) located in the LHA. Immunoreactivity for c-Fos (black) was observed throughout the brain. Arrowheads indicate
c-Fos- and orexin-double positive neurons (arrowheads). Arrows indicate c-Fos-negative and orexin-positive neurons. Insets are higher magnifications of the squared region.
Scale bars 100 ␮m. (B) Bar graph summarizing the data in (A) shows the expression rate of c-Fos in orexin neurons. The black bar indicates the condition without light
illumination and the green bar indicates the condition with 1 h light illumination before decapitation. Values are represented as means ± S.E.M. *, p < 0.05 vs. control in
orexin-tTA; TetO ArchT transgenic mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
in SWS, accompanied by decreased time spent awake in the
early dark period. Sleep/wakefulness state transition frequency
in control mice was 6.9 ± 1.5 (n = 7), whereas the frequency in
light-illuminated mice was 16.7 ± 3.3 (n = 7, p = 0.019, paired ttest) (Fig. 6Be). However, locomotor activity was not significantly
altered: locomotor activity of control and light-illuminated groups
was 1455.3 ± 210.8 counts (n = 7) and 1087.7 ± 185.4 counts (n = 7,
p = 0.12, NS, paired t-test), respectively (Fig. 6Bd). These results suggest that the increase in the fragmentation of sleep/wakefulness
cycles, which represents the dominant clinical manifestation of
narcolepsy, is modeled by the inability to maintain consolidated
wakefulness during light illumination in orexin-tTA; TetO ArchT
mice.
Next, we examined the effect of a 1 h light illumination during
the light period (12:00–13:00) (Fig. 6C). Mice are nocturnal animals,
and are largely inactive in the light period. They spend most of their
time sleeping during the light period (Fig. 6Ca). Green light illumination during the light period did not affect sleep/wakefulness
patterns. They showed almost the same sleep/wakefulness patterns as control mice (Fig. 6Ca and Cb). There were no significant
differences in total times of sleep/wakefulness states between
light-illumination and no-illumination control groups (Fig. 6Cc).
Without photic illumination, the total time in wakefulness, SWS
and REM sleep was 12.2 ± 4.3 min (n = 5), 44.5 ± 4.3 min (n = 5) and
3.4 ± 0.5 min (n = 5), respectively. Following green light illumination for 1 h, the total time in wakefulness, SWS and REM sleep
was 14.8 ± 6.5 min (n = 5, p = 0.64, NS, paired t-test), 43.2 ± 5.7 min
(n = 5, p = 0.80, NS, paired t-test) and 2.1 ± 1.3 min (n = 5, p = 0.37, NS,
paired t-test), respectively. In accordance with these results, locomotor activity and behavioral state transition frequency was not
significantly different between groups (n = 5, Fig. 6Cd and Ce). These
results suggest that inhibition of orexin neuron activity induced
fragmentation of sleep/wakefulness in the dark period, but not in
the light period.
4. Discussion
4.1. TetO ArchT mice are useful for sustained inhibition in vivo
Optogenetics is a powerful tool to study the regulatory mechanisms of behavior. Although activation of neurons using ChR2 often
succeeded in controlling an animal’s behavior, few studies have
succeed in modifying behavior by silencing neurons. Previously,
we used halorhodopsin (HaloR) to inhibit the activity of orexin
neurons and found that inhibition of orexin neurons induced SWS
when the mice were awake [35]. However, silencing through HaloR
was insufficient to completely inhibit the activity of neurons and
it was difficult to inhibit activity for more than 1 min, likely due to
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T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
Fig. 6. Green light illumination increases time spent in SWS during the dark period. (A) Illustration showing the timing of the 1 h continuous illumination in vivo. Orexin-tTA;
TetO ArchT mice were housed under controlled lighting conditions (12 h light/dark cycle; light on 8:00–20:00). These mice were illuminated during 20:00–21:00 (dark period)
or during 12:00–13:00 (light period). Each mouse received photic illumination with an interval of at least 2 days between each illumination. (B) The results of 1 h green
light illumination from 20:00 to 21:00. Shown are representative 2 h (19:30–21:30) hypnograms for the basal condition (Ba) and after a 1 h light illumination (Bb) of an
orexin-tTA; TetO ArchT transgenic mouse during the dark period. Hypnograms were obtained by simultaneous EEG/EMG recording. Green light was applied through plastic
fiber optics (diameter of 0.5 mm) that were bilaterally inserted into the hypothalamus. The green bar indicates the duration of light illumination. (Bc, Bd and Be) Bar graphs
summarizing the data in (Ba) and (Bb): (Bc) total time spent in each vigilance state; (Bd) locomotor activity; (Be) the number of state transitions. (C) The results of a 1 h green
light illumination from 12:00 to 13:00. Representative 2 h (11:30–13:30) hypnograms for the basal condition (Ca) and after 1 h light illumination (Cb) of an orexin-tTA; TetO
ArchT transgenic mouse during the light period. (Cc, Cd and Ce) Bar graphs summarizing the data in (Ca) and (Cb): (Cc) total time spent in each vigilance state; (Cd) locomotor
activity; (Ce) number of state transitions. Wake; wakefulness. Light; Light illumination. Values are represented as means ± S.E.M. *, p < 0.05 vs. control in orexin-tTA; TetO
ArchT transgenic mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
desensitization mechanisms, at least in orexin neurons. To control
instinctive behavior, such as sleep/wakefulness state in mice, it is
essential to manipulate the activity of neurons for longer periods.
ArchT, a green light-driven proton pump, produced strong and long
lasting inhibition compared with HaloR. Prolonged manipulation
of orexin neuronal activity seems vital to understand the neuronal
circuitry underlying the maintenance of sleep/wakefulness in addition to the initiation of sleep/wakefulness.
In the present study, we generated a new line of transgenic mice,
TetO ArchT BAC transgenic mice, which enable expression of ArchT
in a specific cell type following breeding with tTA mice, in which
a specific promoter drives tTA expression. We used the Tet system based on our previous report that this system can amplify
the expression levels of transgene compared to direct expression using cell-type specific promoters [37]. High expression levels
of light-activated proteins are important to achieve a significant
manipulation of membrane potential, particularly in silencing neurons. The utility of using the Tet transgenic system for optogenetic
manipulation was exemplified by the KENGE-tet system, employing TetO ChR2 (C128S) mice. The specific expression of ChR2 (C128S)
was observed in Ca2+ -calmodulin-dependent protein kinase II
(␣CaMKII)-expressing neurons, 5-hydroxytryptamine receptor 5B
(Htr5B)-expressing neurons, GABAergic neurons, astrocytes, oligodendrocytes and microglia when crossing with ˛CaMKII-tTA mice,
Htr5B-tTA mice, GAD67-tTA mice, Mlc1-tTA mice, PLP-tTA mice and
Iba1-tTA mice, respectively [37]. The generation of TetO ArchT mice
further expands the researchers toolbox to inhibit various types of
cells. To demonstrate the usefulness of TetO ArchT BAC transgenic
mice, these animals were bred with orexin-tTA mice. Immunohistochemical data revealed that ArchT was expressed in more than 70%
of orexin neurons with no ectopic expression in non-orexin neurons in the orexin-tTA; TetO ArchT double-transgenic mouse brain.
In vitro slice patch clamp recordings demonstrated that green
light illumination instantaneously induced hyperpolarization or
outward current in ArchT-expressing orexin neurons repetitively
and in a light intensity-dependent manner. Silencing of orexin
neurons was further confirmed by using a loose cell-attached
recording, which records firing frequency without affecting intracellular conditions of the recorded neuron. In this recording
condition, green light illumination for 10 min enabled a complete silencing of ArchT-expressing orexin neurons, suggesting a
probability of inhibiting activity of orexin neurons in vivo. This
implication was strongly supported by our immunohistochemical data for c-Fos, a marker of neuronal activity. Continuous 1 h
green light illumination from the start of the dark period significantly decreased the number of orexin neurons expressing c-Fos,
suggesting that orexin neuronal activity was inhibited in vivo. These
results indicate that ArchT is a more potent silencer for long-lasting
manipulation compared with HaloR, at least in orexin neurons.
4.2. Inhibition of orexin neuronal activity increases time spent in
SWS only during the dark period
In this study, we demonstrated that acute (1 min) optogenetic
inhibition of orexin neurons through ArchT increased transit probability from wakefulness to SWS in the light period, but failed
in the dark period, in good agreement with our previous report
using HaloR [35]. In contrast, the present study showed that 1 h
optogenetic inhibition of orexin neurons through ArchT increased
time spent in SWS and decreased time spent in wakefulness in
T. Tsunematsu et al. / Behavioural Brain Research 255 (2013) 64–74
the dark period, but showed little effect on sleep/wakefulness
in the light period. Why does this differential result arise? One
possibility is that HaloR-induced inhibition was too weak to
completely block the activity of orexin neurons during the dark
period. Currents induced by ArchT (sustained current) are 20
times greater than those mediated by HaloR in orexin neurons.
ArchT might completely inhibit the activity of orexin neurons
even in the early dark period when orexin neurons are supposed
to receive many excitatory inputs and to be highly active. The
other possible factor is length of inhibition time. Likely due to
desensitization mechanisms, HaloR could only produce inhibition
for 1 min. Inhibition for such a limited period might be insufficient to induce sleep in the active period since a 1 min inhibition
of orexin neurons during the dark period also had little effect
on sleep/wakefulness patterns in orexin/Archaerhodopsin-3(Arch)
transgenic mice. Orexin/Arch mice directly drive Arch expression
in orexin neurons using a prepro-orexin promoter and inhibit
orexin neurons with strength comparable to HaloR (unpublished
data). Previous studies reported that CSF orexin levels in rats display a high peak during the dark period and lowest levels during
the light period [40]. Therefore, 1 min silencing might not affect
sleep/wakefulness patterns since orexin peptide concentrations are
high in the CSF in the dark period. Comparatively, 1 h silencing of
orexin neurons during the light period had little effect. This result
is in agreement with previous reports demonstrating that orexin
neuronal activity is necessary for the maintenance of wakefulness
states rather than generating a waking state since orexin neuronablated mice and human narcolepsy patients have impairments in
maintaining wakefulness but they can awaken. Mice are nocturnal animals that spend most of their time sleeping during the light
period, but also frequently show brief waking episodes similar to a
fragmentation of sleep/wakefulness. Thus, silencing for 1 h might
not have induced further fragmentation within the light period.
The present study revealed that 1 h continuous optogenetic
illumination in the dark period induced a fragmentation of the
sleep/wakefulness cycle similar to mice lacking the orexin gene,
the OX2R gene, orexin neurons and dogs with null mutations
in orexin signaling [3–6]. However, inhibition of orexin neurons
through ArchT did not induce a cataplexy-like behavioral arrest and
direct transition from wakefulness to REM sleep. These behaviors
are observed in prepro-orexin knockout mice and orexin neuronablated mice. These differences in phenotype might be related to
the percentage of ArchT-expressing orexin neurons in our study.
Immunohistochemical data revealed that ArchT was expressed
in 72% of orexin neurons. In accordance with this result, 13% of
orexin neurons still expressed c-Fos in the dark period even when
orexin neurons were inhibited by green light illumination in orexintTA; TetO ArchT mice. These results indicate that activation of a
small number of orexin neurons might be sufficient to protect a
direct transition from wakefulness to REM sleep and cataplexylike behavior. Another possibility is that reconstruction of neuronal
networks after the loss of orexin neurons or loss of orexin signal
might be necessary to trigger these symptoms. Similar results are
observed in rats, dogs and humans that are orally administered
the dual orexin receptor (OX1R and OX2R) antagonist, almorexant [41]. Pharmacological blockage of orexin receptors during the
active period increased SWS and REM sleep and decreased wakefulness without evidence of cataplectic symptoms or direct transition
from wakefulness to REM sleep.
5. Conclusions
In this study, we generated a novel line of TetO ArchT BAC transgenic mice. Data from Orexin-tTA; TetO ArchT double-transgenic
mice confirmed that ArchT is correctly expressed and functions to
73
inhibit the activity of tTA-expressing neurons. Long-lasting silencing of orexin neurons in vivo increased time spent in SWS and
decreased time spent awake during the dark period but not during
the light period, indicating the physiological importance of orexin
neuronal activity in the maintenance of arousal during the active
phase.
Acknowledgements
This study was supported by the JST PRESTO program, Grantin-Aid for Scientific Research on Innovative Areas “Mesoscopic
Neurocircuitry” (23115103), Grant-in-Aid for Scientific Research
(B) (23300142) (A.Y.), a Japan Society for Promotion of Science
postdoctoral fellowship (T.T.).We thank Dr. A. Inutsuka for confocal microscopic observation, C. Saito and K. Nishimura for technical
assistance.
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